The present invention relates to a human novel gene and in particular to a gene for a novel transmembrane protein considered to be a pathogenic gene for bipolar affective disorder.
Bipolar affective disorder is a disease occurring mainly at an age of about twenty years old, to the middle age and constitutes one of two major endogenous psychoses along with schizophrenia. The center of its mental symptoms is emotional impairment, and this disease is characterized that even after the morbid phase occurs repeatedly during a periodic course, the patient is restored to normal mental conditions without having mental ruin (impaired conditions). The morbid phase includes a manic state showing high emotion and promoted willingness and a depressive state of emotion, and each morbid phase (for a few weeks to a few months) repeatedly appears or both the morbid phases appear alternately, but in some cases the morbid phase may end only once. For treatment of this disease, an anti-depressant or an anti-psychosis medicine is used, and by the advent of lithium salts, chemotherapy has occupied the most important position.
The morbidity risk factor of this disease is estimated to be approximately 0.4% of the population, but in consideration in mild cases not necessitating medical treatment, the morbidity may considerably exceed this figure. The morbidity according to a fact-finding survey conducted in 1963 by the Ministry of Health and Welfare was 0.02%, but naturally this morbidity does not cover non-manifested cases at the time of the survey. In the incidence of this disease, there is a certain difference depending on human race, differentiation and region, and it is noted that the incidence tends to be high in highly differentiated races or in upper classes. It is said that the number of female patients is higher than male patients, but this is necessarily not evident in Japan.
This disease is classified into a unipolar type (type showing the depressive phase only) and a bipolar type (type showing both the phases or the manic phase only), and comparison between the unipolar type and the bipolar type indicates that the incidence of the unipolar type is considerably higher at a ratio of 1:6 to 10. In particular, this trend is significant at the middle age or thereafter.
Diagnosis of bipolar affective disorder is made according to clinical symptoms based on emotional impairment. There is also a report that the level of serotonin or noradrenaline is varied with emotional impairment, and particular attention is paid to the movement of monoamine metabolism-related substances in blood, urine, or cerebrospinal fluid. Further, attention is also paid to impairments in the endocrine functions of the thyroid gland, adrenal cortex etc. and abnormalities in the metabolism of electrolytes affecting the mechanism of neurotransmission, but there is no established method for clinical examination utilizable for diagnosis thereof.
It is considered that a genetic predisposition plays an important role in the onset of bipolar affective disorder. According to a lineage study (empirical hereditary prognosis), the morbidity risk factor of a patient""s family is significantly higher than ordinary persons. The morbidity in such a family is estimated to be nearly 10 to 15% and there is no significant difference among patient""s brothers, parents and children. According to a recent study, however, bipolar affective disorder is genetically not necessarily a single unit, and the morbidity risk factor of bipolar bipolar affective disorder in the family is genetically higher than that of unipolar depressive insanity in the family, and it is pointed out that generally, in a family of a patient with the bipolar type, the percentage of patients with the bipolar type in the family is relatively high, while in a family of a patient with the unipolar type, the percentage of patients with the unipolar type in the family is relatively high.
In study by a twins method, the involvement of a genetic predisposition is evident, and the morbidity of the disease in both one-egg twins is significantly higher than in both two-egg twins. However, because there is not few exceptional cases, it is considered that the expression of a gene for bipolar affective disorder is considerably affected by the environment as is the case with a gene for schizophrenia [1].
The hereditary mode of bipolar affective disorder, excluding a limited number of families having considerably negative factors in the family, cannot be elucidated in terms of the Mendelian heredity concerned with a single gene, so it is considered that the majority of cases are due to polyfactor-polygene heredity in which a plurality of genes and environmental factors are involved. Further, endogenous factors in bipolar affective disorder, that is, disease-specific changes in organs, biological markers, onset mechanism etc. are not revealed, thus making genetic study further difficult.
In 1987, Egeland et al. reported that there is a strong linkage between chromosome 11q15 HRAS1 and INS genes and bipolar affective disorder [2]. At the same time, in a study where color blindness in the chromosome Xq28 and GP6D were used as markers in a family liable to bipolar impairment, Baron et al. reported that there is a strong linkage thereof to bipolar impairment [3]. However, these and later reports showed some results denying the linkage, and the above study is not an established finding. Besides, regions having a high linkage to bipolar affective disorder include Xq29-27 [4], 4q16 [5], 18q22-23 [6], 18centro [7] and 21q22.3 [8].
However, in the linkage study of bipolar affective disorder, a large number of findings suggesting the linkage are obtained while there are many results denying these findings [9] so that a state of confusion continues. As a result, none of these findings are established, and at present, there is no cloned gene for bipolar affective disorder [10].
The problem to be solved by the invention is to determine the nucleotide sequence of a pathogenic gene for bipolar affective disorder as well as the amino acid sequence of a protein encoded by said nucleotide sequence.
As a result of their eager study for solving the problem described above, the present inventors isolated a new gene from human chromosome 21q22.3 region and determined its nucleotide sequence. Genes from patients with bipolar affective disorder were amplified by PCR using primers set up on the basis of said nucleotide sequence and analyzed, and as a result it was confirmed that there was a deletion in exon 5 in 10 samples out of 20 samples from patients with bipolar bipolar affective disorder. Accordingly, it is considered that this gene is a pathogenic gene for bipolar affective disorder.
It was revealed that the open reading frame of this gene designated TRPC7 codes for a protein with a molecular weight of 171,217 consisting of 1,503 residues having 7 transmembrane regions. Further, as a result of a search for homology in a database, said protein showed homology with Drosophila TRP (transient receptor potential) protein and human TRP protein as calcium channels.
An object of the present invention is to provide a polypeptide particularly a polypeptide shown in SEQ ID NO:2, which was designated TRPC7 because of its homology with known amino acid sequences such as Drosophila TRP protein and human TRP protein. TRPC7 has seven transmembrane regions similar to other TRPs and is estimated to act as a calcium channel.
A further object of the present invention is to provide a polynucleotide coding for TRPC7, in particular a polynucleotide coding for the polypeptide designated TRPC7 in the present specification. A particularly preferable example of this mode of the present invention is a polynucleotide comprising a region coding for human TRPC7 in the sequence shown in SEQ ID NO:1.
According to this mode of this invention, there are provided isolated nucleic acid molecules coding for human TRPC7, including mRNA, cDNA, genomic DNA and fragments thereof, and in a further specific example of this mode, there are provided biologically, diagnostically, clinically or therapeutically useful fragments thereof, including varieties, analogues or derivatives thereof or fragments of the varieties, analogues or derivatives. A particularly preferable example of this mode of this invention is varieties of a natural allele of human TRPC7.
An object of this invention is to provide a TRPC7 polypeptide in particular a human TRPC7 polypeptide which can be used in treatment of bipolar affective disorder. According to this mode of this invention, there is provided a novel human-derived polypeptide designated TRPC7 in this specification, as well as biologically, diagnostically or therapeutically useful fragments, varieties and derivatives thereof, varieties and derivatives of these fragments, and analogues thereof. A particularly preferable example of this mode according to this invention is varieties of human TRPC7 encoded by a natural allele of human TRPC7 gene. According to another mode of this invention, there is provided a method of screening a compound binding to the polypeptide of the present invention to activate or inhibit it.
A further object of this invention is to provide a method of producing said polypeptide, polypeptide fragments, varieties and derivatives, fragments of the varieties and derivatives, and analogues thereof. According to a preferable example of this mode, there is provided a method of producing said TRPC7 polypeptide, wherein host cells containing a human TRPC7-coding nucleotide sequence integrated therein and induced extraneously to be expressible are cultured under conditions for expression of human TRPC7 in the host and the polypeptide thus expressed is recovered.
According to a further other object of this invention, there are provided a product, a composition, a process and a method for utilizing said polypeptide and polynucleotide in particular for research, biological, clinical and therapeutic purposes.
According to a preferable example of this mode of the present invention, there are particularly provided a product, a composition and a method for the following purposes: evaluation of TRPC7 expression in cells by measuring the TRPC7 polypeptide or TRPC7 mRNA; treatment in vitro, ex vivo or in vivo of bipolar affective disorder by exposing cells to the TRPC7 polypeptide or polynucleotide disclosed in this specification; analysis of genetic deformation and defects such as deletions in TRPC7 gene; and increase of TRPC7 function or improvement of TRPC7 malfunction by administering the TRPC7 polypeptide or polynucleotide to creatures. According to a other embodiment of this invention, there is provided a method of using an active compound for stimulating the receptor polypeptide of this invention, to treat symptoms related to deficient expression of TRPC7. According to a still other embodiment of this invention, there is provided a method of using an inhibitory compound for treatment of symptoms accompanying excessive expression of TRPC7.
According to a further other mode of this invention, there are provided synthetic or recombinant TRPC7 polypeptide, conservatively substituted polypeptides thereof and derivatives thereof, an antibody and an anti-idiotype antibody against the same, which are used in diagnosis, therapy and/or research.
A further other object of this invention is to provide synthetic, isolated or recombinant polypeptides designed as subtype to inhibit or mimic TRPC7 or various fragments thereof. According to these examples or other examples of a further other mode of this invention, a probe hybridizing with the human TRPC7 sequence is provided. In a further preferable mode of this invention, an antibody against TRPC7 polypeptide is provided. In a very preferable example in this respect, the antibody is very selective against human TRPC7. According to another mode of this invention, TRPC7 agonist is provided. A preferable agonist is a molecule mimicking TRPC7, a TRPC7-binding molecule, or a molecule inducing or increasing TRPC7-inducing response. Further, a preferable agonist is a molecule which interacts with TRPC7, TRPC7 polypeptides or other modulators for TRPC7 activity thereby activating or increasing one or more effects of TRPC7. According to another mode of this invention, TRPC7 antagonist is provided. A preferable antagonist mimics TRPC7, thus binding to a TRPC7-binding molecule, but does not induce one or more responses for inducing TRPC7. Further, a preferable antagonist is a molecule binding to TRPC7 or interacting with it to inhibit one or more effects of TRPC7 or to prevent expression of TRPC7. According to an additional mode of this invention, there is provided a TRPC7 polynucleotide or a composition comprising the TRPC7 polynucleotide, which is administered into cells in vitro, ex vivo and in vivo or into multicellular creatures. In a particular preferable example in this mode of the present invention, said composition comprises the TRPC7 polynucleotide for expressing TRPC7 polypeptide in a host for treatment of the disease. Its expression in a patient with bipolar affective disorder is particularly preferable in this respect.
Other objects, features, advantages and modes of the present invention will be obvious to those skilled in the art in view of the following description of this specification. However, the description and the examples below are preferable modes of this invention and are shown merely for illustrative purposes. From the following description and other disclosure of this specification, it would be evident to those skilled in the art that various alternations and modifications are possible within the scope described in this specification. The drawings also show merely illustrative examples of this invention, and are not intended to limit the disclosure of the invention disclosed in this specification.